Detection of par in the csf of patients with parkinson&#39;s disease

ABSTRACT

Poly(ADP-ribose) (PAR) is a protein implicated in numerous neurodegenerative disease states including Parkinson&#39;s disease (PD). To date, no routine laboratory test has been developed to diagnosis, assess or monitor patients suffering from PD. Disclosed herein a novel method to assess the PAR concentration in the cerebral spinal fluid (CSF) of a patient and correlate that concentration to the medical condition of a patient with PD. Also disclosed is the use of PAR as a biomarker for PD.

This application claims priority to U.S. Provisional Application62/514,316 filed on Jun. 2, 2017 and U.S. Provisional Application62/679,161 filed on Jun. 1, 2018, both of which are incorporated byreference in their entirety for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH & DEVELOPMENT

This invention was made with government support under grant no. NS38377and U01NS082133 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

Poly(ADP-ribose) (“PAR”) polymerase-1 (“PARP-1”) is an important nuclearenzyme that responds to DNA damage and is required for DNA repair. Uponactivation, PARP-1 catalyzes the transfer of ADP-ribose fromnicotinamide adenine dinucleotide (“NAD+”) and conjugates PAR onto avariety of nuclear proteins such as histones, DNA polymerases,topoisomerases, and transcription factors as well as auto-modificationof PARP-1 itself, thus regulating a variety of physiologic processes.Excessive activation of PARP-1 leads to an intrinsic cell death program,which has been designated parthanatos (or, alternatively,PARP-1-dependent cell death) to distinguish it from necrosis andapoptosis. Parthanatos is known to occur in many diseases and conditionssuch as stroke, Parkinson's disease, heart attack, diabetes, andischemia reperfusion injury. PARP-1 inhibition or PARP-1 gene deletionis markedly protective in models of many cell injury paradigms,including stroke, trauma, ischemia-reperfusion injury, diabetes, andneurodegenerative diseases, indicating that parthanatos plays aprominent role in these disorders.

The mitochondrial protein apoptosis-inducing factor (“AIF”) plays apivotal role in parthanatos, during which AIF is released from themitochondria and translocates to the nucleus. AIF is a mitochondrialoxidoreductase that, like cytochrome C, has two independent functions.The first is within mitochondria, involving cell survival, thought to bethrough assembly or stabilization of respiratory complex I. The secondis as a promoter of parthanatos cell death. AIF is released into thecytoplasm following PARP-1 activation, ultimately entering the nucleusto induce cell death.

Parkinson's disease (PD) is an age-related neurodegenerative disease inwhich α-syn deposits as fibrils in intracytoplasmic inclusions instructures termed Lewy bodies and neurites. Recombinant α-syn can beaggregated in vitro to form fibrils similar in structure to those foundin vivo, and these α-syn pre-formed fibrils (α-syn PFF) can spread in aprion-like manner: both in in vitro neuronal cultures and in vivo wheninjected into the mouse brain with accompanying phosphorylation of α-synon serine 129, a marker of pathologic α-syn and neurotoxicity. While itis clear that aggregated α-syn underlies the pathology of PD, whatdrives abnormal aggregation of α-syn as well as the cell injury anddeath mechanisms that are activated by this aggregation are not yetknown. Since poly (ADP-ribose) (PAR) polymerase-1 (PARP-1) and PAR playa major contributing role in cell death relevant to neurologicdisorders, here the role for PARP-1 and PAR in pathologic α-syn inducedneurodegeneration was evaluated.

Additionally, PD is a slowly progressive, neurodegenerative CNS disordercharacterized by slow and decreased movement, muscular rigidity, restingtremor, postural instability, cognitive impairment and dementia. Themajor pathological feature of PD is selective degeneration ofdopaminergic neurons in the substantia nigra pars compacta (SNpc) andloss of their terminals in the caudate and putamen. Loss of substantianigra neurons, which project into the caudate nucleus and putamen,depletes dopamine in these areas. Evidence suggests that multiplefactors, including genetic and environmental ones, contribute to thedopaminergic neurodegeneration in this neurodegenerative disease.

The medical treatment of PD is directed to stopping, slowing or reducingthe extent of or minimizing the neurodegenerative process innigrostriatal neurons (neuroprotective therapy) and eliminating thebiochemical imbalance (symptomatic therapy). The main directions ofsymptomatic therapy in PD are to increase dopamine synthesis, orstimulate dopamine receptors activity and dopamine release from thepresynaptic space, and to inhibit dopamine reuptake by presynapticreceptors and dopamine catabolism.

Because there is no known cure for PD at this time and differentpatients respond to treatment methods differently, it is important for amedical professional to carefully monitor disease progression in apatient. This allows the medical professional to adjust or alter themedical treatment the patient receives in the event that the currenttreatment is ineffective. The current method for monitoring PD progressis a subjective scale based on the level of disability and impairmentexperienced by a patient.

Thus there remains a need to more specifically to assess and monitor thetreatment and progression of PD patients in order fine tune medicaltreatment. A need also exists for a quantitative as well as qualitativescale that eliminates or reduces the subjective component of theassessment.

BRIEF DESCRIPTION OF THE INVENTION

In a first aspect, disclosed herein is a method for determining apoly(ADP-ribose) (PAR) concentration in cerebral spinal fluid, themethod comprising collecting a sample of cerebrospinal fluid (CSF) froma patient, performing a PAR-sandwich ELISA on the CSF sample, therebydetermining the PAR concentration in the CSF.

In a second aspect, disclosed herein is a method for determining thetherapeutic efficacy of a medical treatment for Parkinson's disease, themethod comprising collecting a sample of cerebrospinal fluid (CSF) froma patient, measuring a poly(ADP-ribose) (PAR) concentration in the CSFsample, and comparing the PAR concentration in the patient to a PARconcentration in at least one control sample.

In yet another aspect, disclosed herein is a method for monitoring thedisease progression of a patient with Parkinson's disease (PD), themethod comprising collecting a sample of cerebrospinal fluid (CSF) froma patient, measuring a poly(ADP-ribose) (PAR) concentration in the CSFsample, and comparing the PAR concentration in the patient to a PARconcentration in at least one control sample, wherein the patient isreceiving at least one medical treatment for PD.

In still yet another aspect, disclosed herein is a method of diagnosinga patient with Parkinson's disease, the method comprising collecting asample of cerebrospinal fluid (CSF) from a patient, measuring apoly(ADP-ribose) (PAR) concentration in the CSF sample, and comparingthe PAR concentration in the patient to a PAR concentration in at leastone control sample.

In still yet another aspect, disclosed herein is a theranostic methodfor Parkinson's disease, the method comprising collecting a sample ofcerebrospinal fluid (CSF) from a patient who is receiving at least onemedical treatment for PD, measuring a poly(ADP-ribose) (PAR)concentration in the CSF sample, comparing the PAR concentration in thepatient to a PAR concentration in at least one control sample.

In still yet another aspect, disclosed herein is the use of PAR in theCSF of a patient as a biomarker for PD.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of the processes in a cell illustratingthe model of PAR-dependent AIF release in parthanatos.

FIG. 2 is a schematic drawing of the sandwich ELISA assay with ananti-PAR antibody and a concentration curve.

FIG. 3 is a bar graph of the concentration of PAR in the CSF of a PDpatient versus healthy controls.

FIG. 4 is a bar graph of the concentration of PAR in PD-normal patientsvs PD-cognitively impaired patients.

FIG. 5 shows activation of PARP-1 in α-syn PFF-treated primary corticalneurons. The representative western blot analysis (top) andquantification (bottom) of the levels of PAR accumulation. Barsrepresent mean±s.e.m. One-way ANOVA followed by Tukey's post hoc test(n=3-4).

FIG. 6 shows representative images of Hoechst and propidium iodide (PI)staining from primary cortical neurons pre-incubated with either ABT-888(10 μM), AG-014699 (1 μM) or BMN 673 (10 μM) for 1 h, and furtherincubated with α-syn PFF (5 μg/ml) for 14 days. Scale bar, 20 μm.

FIG. 7 shows quantification of cell death. Bars represent mean±s.e.m.One-way ANOVA followed by Tukey's post hoc test (n=3).

FIG. 8 shows inhibition of α-syn PFF-induced PAR accumulation wasdetermined by western blot analysis.

FIG. 9 shows representative images of Hoechst and propidium iodide (PI)staining from primary cortical neurons transduced with AAV-sgCon orAAV-sgPARP-1, and further incubated with α-syn PFF for 14 days. Scalebar, 20 μm.

FIG. 10 shows quantification of cell death. Bars represent mean±s.e.m.(n=3).

FIG. 11 shows representative images of Hoechst and propidium iodide (PI)staining from WT or PARP-1 KO primary cortical neurons, and furtherincubated with α-syn PFF for 14 days. Scale bar, 20 μm.

FIG. 12 shows quantification of cell death. Bars represent mean±s.e.m.One-way ANOVA followed by Tukey's post hoc test (n=3). *P<0.05,**P<0.005, ***P<0.0005.

FIG. 13 shows representative immunoblots and quantification of thelevels of PAR accumulation in the striatum of α-syn PFF injected mice.Bars represent mean±s.e.m. One-way ANOVA followed by Tukey's post hoctest (n=4).

FIG. 14 shows representative TH and Nissl staining of SNpc DA neurons ofα-syn PFF injected WT, PARP-1KO, and WT mice fed with ABT-888 at 6months after intrastriatal α-syn PFF or PBS injection.

FIG. 15 shows stereological counts. Data are mean±s.e.m. One-way ANOVAfollowed by Tukey's post hoc test (n=5 to 7 mice per group).

FIG. 16 shows DA concentrations in the striatum of WT, PARP-1 KO, and WTmice fed with ABT-888 at 6 months after intrastriatal α-syn PFF or PBSinjection measured by HPLC. Bars represent mean±s.e.m. One-way ANOVAfollowed by Tukey's post hoc test. (n=6 to 30 mice per group).

FIG. 17 shows pole test results 180 days after α-syn PFF injection. Thepole test was performed in WT, PARP-1 KO, or WT mice fed with ABT-888.Data are the means s.e.m. One-way ANOVA followed by Tukey's post hoctest (n=7 to 9 mice per group). *P<0.05, **P<0.005, ***P<0.001.

FIG. 18 shows grip strength results 180 days after α-syn PFF injection.The grip strength test was performed in WT, PARP-1 KO, or WT mice fedwith ABT-888. Data are the means±s.e.m. One-way ANOVA followed byTukey's post hoc test (n=7 to 9 mice per group). *P<0.05, **P<0.005,***P<0.001.

FIG. 19 shows acceleration of α-syn fibrillization by PAR. Monomericα-syn either with or without 5 nM purified PAR was incubated at 37° C.for indicated times. Fibrillization of α-syn was detected byimmunoblotting using α-syn antibody (top). Data are mean±s.e.m.(bottom). One-way ANOVA followed by Tukey's post hoc test (n=3).

FIG. 20 shows the rate of formation of α-syn fibrils either with orwithout PAR was monitored by thioflavin T fluorescence (n=3).

FIG. 21 shows representative transmission electron microscopy (TEM)images for α-syn fibrils. Scale bar, 200 nm.

FIG. 22 shows suppression of NMDA-induced α-syn fibrillization in PARP-1KO neurons. Primary cortical neurons from WT or PARP-1 KO embryos weretransduced with AAV-α-syn and then further incubated with 500 μM NMDAfor 5 min. The α-syn fibrillization was detected by western blotanalysis 6 h after NMDA treatment.

FIG. 23 shows prevention of NMDA-induced α-syn fibrillization by PARP1inhibitors. The primary cortical neurons transduced with AAV-α-syn werepre-treated with 10 μM ABT-888 or 1 μM AG-014699 for 1 h, and furtherincubated with 500 μM NMDA for 5 min. The α-syn fibrillization wasdetected by western blot analysis 6 h after NMDA treatment.

FIG. 24 shows α-syn PFF or PAR-α-syn PFF incubated with increasingconcentration of PK (0-2.5 μg/ml) and immunoblotted with α-syn antibody(top). Quantification represents the ratio of cleaved to uncleaved α-syn(bottom). Data are mean±s.e.m. (bottom). One-way ANOVA followed byTukey's post hoc test (n=3).

FIG. 25 shows representative immunostaining of p-α-syn (red) in primarycortical neurons treated with α-syn PFF or PAR-α-syn PFF for 1, 4 and 7days. Quantification of p-α-syn signals normalized with DAPI (right).Bars represent mean±s.e.m. (bottom). One-way ANOVA followed by Tukey'spost hoc test (n=5).

FIG. 26 shows primary cortical neurons treated with α-syn PFF orPAR-α-syn PFF were sequentially extracted with 1% TX-100 (TX soluble)and 2% SDS (TX insoluble). Lysates were subjected to immunoblottingusing α-syn, p-α-syn, and GAPDH antibodies. Bars represent mean±s.e.m.One-way ANOVA followed by Tukey's post hoc test (n=3). ND, not detected.*P<0.05, **P<0.005, ***P<0.0005 as compared to α-syn PFF for 1 day.^(#)P<0.05, ^(##)P<0.005, ^(###)P<0.0005.

FIG. 27 shows representative TH and Nissl staining of SNpc DA neurons ofWT mice at 1, 3, and 6 months after instrastriatal PBS, α-syn PFF,PAR-α-syn PFF, or PAR injection.

FIG. 28 shows stereological counts. Bars represent mean±s.e.m. One-wayANOVA followed by Tukey's post hoc test (n=5 to 8 mice per group).

FIG. 29 shows DA concentrations in the striatum of PBS, α-syn PFF,PAR-α-syn PFF, or PAR-injected mice at 1, 3, and 6 months measured byHPLC. Bars represent mean±s.e.m. One-way ANOVA followed by Tukey's posthoc test (n=4 to 6 mice per group).

FIG. 30 shows representative p-α-syn immunostaining in the SNpc of WTmice at 1, 3, and 6 months after instrastriatal PBS, α-syn PFF,PAR-α-syn PFF, or PAR injection. Scale bar, 100 μm.

FIG. 31 shows quantification of p-α-syn levels. Bars representmean±s.e.m. One-way ANOVA followed by Tukey's post hoc test (n=5 to 8mice per group).

FIG. 32 shows behavioral abnormalities of PBS, α-syn PFF, PAR-α-syn PFF,or PAR-injected mice at 1, 3, and 6 months measured by pole test. Dataare the means s.e.m. One-way ANOVA followed by Tukey's post hoc test(n=9 to 14 mice per group).

FIG. 33 shows behavioral abnormalities of PBS, α-syn PFF, PAR-α-syn PFF,or PAR-injected mice at 1, 3, and 6 months measured by grip strengthtest. Data are the means s.e.m. One-way ANOVA followed by Tukey's posthoc test (n=9 to 14 mice per group).

FIG. 34 shows increase of PAR in CSF of PD patients. The levels of PARin CSF of healthy controls (n=31) and PD patients (n=80) were determinedby PAR ELISA. Bars represent mean±s.e.m. Student t test with Welch'scorrection.

FIG. 35 shows increase of PAR in CSF of PD patients. The levels of PARin CSF of healthy controls (n=33) and PD patients (n=21) were determinedby PAR ELISA. Bars represent mean±s.e.m. Student t test with Welch'scorrection.

FIG. 36 shows increase and co-localization of PAR in Lewy body of PDpatients. Representative α-syn (red) and PAR (green) immunostaining inthe SNpc of PD patients.

FIG. 37 shows representative images of p-α-syn (red) in primary corticalneurons pre-incubated with either ABT-888 (10 μM), AG-014699 (1 μM) orBMN 673 (10 μM) for 1 h, and further incubated with α-syn PFF for 7days. Nuclei are stained with DAPI (blue).

FIG. 38 shows quantification of p-α-syn signals normalized with DAPI.Bars represent mean±s.e.m. One-way ANOVA followed by Tukey's post hoctest (n=6).

FIG. 39 shows representative immunoblots of α-syn in thedetergent-soluble and insoluble fraction from primary cortical neuronspre-treated with PARP inhibitors for 1 h followed by incubated withα-syn PFF for 7 days.

FIG. 40 shows quantification of α-syn levels in the detergent-insolublefraction normalized to b-actin. Bars represent mean±s.e.m. One-way ANOVAfollowed by Tukey's post hoc test (n=3).

FIG. 41 shows inhibition of α-syn PFF-induced PAR accumulation inprimary cortical neurons infected with AAV-sgPARP1.

FIG. 42 shows inhibition of α-syn PFF-induced PAR accumulation inprimary cortical neurons from PARP1 KO embryos.

FIG. 43 shows representative images of p-α-syn (red) in primary corticalneurons pre-incubated with either ABT-888 (10 μM), AG-014699 (1 μM) orBMN 673 (10 μM) for 1 h, and further incubated with α-syn PFF for 7days. Nuclei are stained with DAPI (blue).

FIG. 44 shows quantification of p-α-syn signals normalized with DAPI.Bars represent mean±s.e.m. One-way ANOVA followed by Tukey's post hoctest (n=6).

FIG. 45 shows representative immunoblots of α-syn in thedetergent-soluble and insoluble fraction from WT or PARP1 KO primarycortical neurons incubated with α-syn PFF for 7 days.

FIG. 46 shows quantification of α-syn levels in the detergent-insolublefraction normalized to b-actin. Bars represent mean±s.e.m. One-way ANOVAfollowed by Tukey's post hoc test (n=3).

FIG. 47 shows representative images of Hoechst and propidium iodide (PI)staining from primary cortical neurons pre-treated with ABT-888, Z-VAD,NEC-1, or 3-MA for 1 h, followed by further incubation with α-syn PFFfor 14 days. Scale bar, 20 μm.

FIG. 48 shows quantification of cell death. Bars represent mean±s.e.m.One-way ANOVA followed by Tukey's post hoc test (n=3). *P<0.05,***P<0.001.

FIG. 49 shows representative images of α-syn PFF transmission. α-syn PFFwas added to chamber 1 (C1) of the microfluidic device. On day 14,p-α-syn was detected in chamber 2 (C2) and chamber 3 (C3) when WTneurons were present in all three chambers, but very limited intensityof p-α-syn in PARP-1 KO neurons in C2 and WT neurons in C3. Scale bar,100 μm.

FIG. 50 shows the p-α-syn signal in high resolution images. Scale bar,10 μm.

FIG. 51 shows quantification of p-α-syn levels in each chamber. Valuesare means±s.e.m. Unpaired student's t test (n=3). *P<0.05, **P<0.005.

FIG. 52 shows representative immunoblots of the midbrain lysates fromWT, PARP-1 KO, and WT mice fed with ABT-888 with misfolded α-syn,p-α-syn, TH, DAT, PAR, and PARP-1 antibodies.

FIG. 53 shows quantification of TH levels. Bars represent mean±s.e.m.One-way ANOVA followed by Tukey's post hoc test (n=3). *P<0.05,**P<0.005, ***P<0.001.

FIG. 54 shows quantification of DAT levels. Bars represent mean±s.e.m.One-way ANOVA followed by Tukey's post hoc test (n=3). *P<0.05,**P<0.005, ***P<0.001.

FIG. 55 shows quantification of insoluble α-syn levels. Bars representmean±s.e.m. One-way ANOVA followed by Tukey's post hoc test (n=3).*P<0.05, **P<0.005, ***P<0.001.

FIG. 56 shows quantification of insoluble p-α-syn levels. Bars representmean±s.e.m. One-way ANOVA followed by Tukey's post hoc test (n=3).*P<0.05, **P<0.005, ***P<0.001.

FIG. 57 shows DA metabolite DOPAC concentrations in the striatum of WT,PARP-1 KO, and WT mice fed with ABT-888 at 6 months after intrastriatalα-syn PFF or PBS injection measured by HPLC. Bars represent mean±s.e.m.One-way ANOVA followed by Tukey's post hoc test. (n=5 to 10 mice pergroup).

FIG. 58 shows DA metabolite 3-MT concentrations in the striatum of WT,PARP-1 KO, and WT mice fed with ABT-888 at 6 months after intrastriatalα-syn PFF or PBS injection measured by HPLC. Bars represent mean±s.e.m.One-way ANOVA followed by Tukey's post hoc test. (n=5 to 10 mice pergroup).

FIG. 59 shows DA metabolite HVA concentrations in the striatum of WT,PARP-1 KO, and WT mice fed with ABT-888 at 6 months after intrastriatalα-syn PFF or PBS injection measured by HPLC. Bars represent mean±s.e.m.One-way ANOVA followed by Tukey's post hoc test. (n=5 to 10 mice pergroup).

FIG. 60 shows representative images of p-α-syn immunostaining from WT,PARP-1 KO, and WT mice fed with ABT-888.

FIG. 61 shows quantification of p-α-syn intensity in amygdala region.Bars represent mean±s.e.m. One-way ANOVA followed by Tukey's post hoctest. (n=4 to 5 mice per group).

FIG. 62 shows quantification of p-α-syn intensity in cortex region. Barsrepresent mean±s.e.m. One-way ANOVA followed by Tukey's post hoc test.(n=4 to 5 mice per group).

FIG. 63 shows quantification of p-α-syn intensity in SNpc region. Barsrepresent mean±s.e.m. One-way ANOVA followed by Tukey's post hoc test.(n=4 to 5 mice per group).

FIG. 64 shows pole test results 180 days after α-syn PFF injection. Thepole test was performed in WT, PARP-1 KO, or WT mice fed with ABT-888.Data are the means s.e.m. One-way ANOVA followed by Tukey's post hoctest (n=6 to 30 mice per group). *P<0.05, **P<0.005, ***P<0.001.

FIG. 65 shows grip strength test results 180 days after α-syn PFFinjection. The grip strength test was performed in WT, PARP-1 KO, or WTmice fed with ABT-888. Data are the means±s.e.m. One-way ANOVA followedby Tukey's post hoc test (n=6 to 30 mice per group). *P<0.05, **P<0.005,***P<0.001.

FIG. 66 shows the effect of temperature on PAR-mediated acceleration ofα-syn fibrillization. Monomeric α-syn either with or without 5 nM PARwas incubated at indicated temperatures for 72 h. Fibrillization ofα-syn was detected by immunoblotting using α-syn antibody.

FIG. 67 shows the concentration dependence of PAR-mediated α-synfibrillization. The α-syn fibrillization was detected by western blotanalysis 36 h after incubation.

FIG. 68 shows quantification of α-syn fibrillization. Bars representmean±s.e.m. One-way ANOVA followed by Tukey's post hoc test.

FIG. 69 shows representative immunoblot of α-syn fibrillizationgenerated by adding 5 nM of PAR, Poly (A), or ADPr at 37° C. for 36 h.

FIG. 70 shows quantification of α-syn fibrillization. Bars representmean±s.e.m. One-way ANOVA followed by Tukey's post hoc test.

FIG. 71 shows primary cortical neurons from WT or PARP1 KO embryos weretransduced with AAV-α-syn for 5 days and then PAR polymer was deliveredwith BioPorter for 6 h. The α-syn fibrillization was detected by westernblot analysis.

FIG. 72 shows WT or PARP1 KO SH-SY5Y cells transfected with α-syn WT for24 h and then further incubated with 50 μM MNNG for 15 min. The α-synfibrillization was detected by western blot analysis 6 h after MNNGtreatment.

FIG. 73 shows WT or PARP1 KO SH-SY5Y cells transfected with α-syn A53Tfor 24 h and then further incubated with 50 μM MNNG for 15 min. Theα-syn fibrillization was detected by western blot analysis 6 h afterMNNG treatment.

FIG. 74 shows WT or PARP1 KO SH-SY5Y cells were transfected with α-synWT for 24 h and then PAR polymer was delivered with BioPorter for 6 h.The α-syn fibrillization was detected by western blot analysis.

FIG. 75 shows SH-SY5Y cells transfected with α-syn WT for 24 h werepretreated with 10 μM ABT-888 or 1 μM AG-014699 for 1 h, and furtherincubated with 50 μM MNNG for 15 min. The α-syn fibrillization wasdetected by western blot analysis 6 h after MNNG treatment. Barsrepresent mean±s.e.m. One-way ANOVA followed by Tukey's post hoc test.*P<0.05, **P<0.005, ***P<0.001.

FIG. 76 shows α-syn PFF or PAR-α-syn PFF was incubated with 0.5 μg/ml ofPK and immunoblotted with epitope-specific antibodies to α-syn.

FIG. 77 shows PAR-α-syn PFF was generated with increasing dose ofpurified PAR polymer. Primary cortical neurons were treated with thesame amount of α-syn PFF or PAR-α-syn PFF for 14 days. Cell death wasdetermined by Hoechst and propidium iodide (PI) staining. Bars representmean±s.e.m. One-way ANOVA followed by Tukey's post hoc test (n=3-4).

FIG. 78 shows representative immunostaining of p-α-syn (red) in primarycortical neurons treated with increasing amount of α-syn PFF orPAR-α-syn PFF for 4 days. Nuclei are stained with DAPI (blue).

FIG. 79 shows quantification of p-α-syn signals normalized with DAPI(n=5). One-way ANOVA with Tukey's post hoc test. *P<0.05, **P<0.005,***P<0.001.

FIG. 80 shows sterological counts of SNpc DA neurons of WT mice at 1, 3,and 6 months after instrastriatal PBS, α-syn PFF, PAR-α-syn PFF, or PARinjection. (A) Ipsilateral Nissl-neurons were counted. Bars representmean±s.e.m. One-way ANOVA followed by Tukey's post hoc test (n=5 to 8mice per group). *P<0.05, ***P<0.001.

FIG. 81 shows sterological counts of SNpc DA neurons of WT mice at 1, 3,and 6 months after instrastriatal PBS, α-syn PFF, PAR-α-syn PFF, or PARinjection. contralateral TH-neurons were counted. Bars representmean±s.e.m. One-way ANOVA followed by Tukey's post hoc test (n=5 to 8mice per group). *P<0.05, ***P<0.001.

FIG. 82 shows sterological counts of SNpc DA neurons of WT mice at 1, 3,and 6 months after instrastriatal PBS, α-syn PFF, PAR-α-syn PFF, or PARinjection. Nissl-positive neurons were counted. Bars representmean±s.e.m. One-way ANOVA followed by Tukey's post hoc test (n=5 to 8mice per group). *P<0.05, ***P<0.001.

FIG. 83 shows a schematic diagram of dopamine metabolism.

FIG. 84 shows no significant difference of DA concentration in thecontralateral striatum of PBS, α-syn PFF, PAR-α-syn PFF, or PAR-injectedmice at 1, 3, and 6 months measured by HPLC. Bars represent mean±s.e.m.One-way ANOVA followed by Tukey's post hoc test.

FIG. 85 shows ipsilateral DOPAC concentration in the striatum of PBS,α-syn PFF, PAR-α-syn PFF, or PAR-injected mice at 1, 3, and 6 monthsmeasured by HPLC. Bars represent mean±s.e.m. One-way ANOVA followed byTukey's post hoc test (n=4 to 6 mice per group). *P<0.05, **P<0.005,***P<0.001.

FIG. 86 shows contralateral DOPAC concentration in the striatum of PBS,α-syn PFF, PAR-α-syn PFF, or PAR-injected mice at 1, 3, and 6 monthsmeasured by HPLC. Bars represent mean±s.e.m. One-way ANOVA followed byTukey's post hoc test (n=4 to 6 mice per group). *P<0.05, **P<0.005,***P<0.001.

FIG. 87 shows ipsilateral 3-MT concentration in the striatum of PBS,α-syn PFF, PAR-α-syn PFF, or PAR-injected mice at 1, 3, and 6 monthsmeasured by HPLC. Bars represent mean±s.e.m. One-way ANOVA followed byTukey's post hoc test (n=4 to 6 mice per group). *P<0.05, **P<0.005,***P<0.001.

FIG. 88 shows contralateral 3-MT concentration in the striatum of PBS,α-syn PFF, PAR-α-syn PFF, or PAR-injected mice at 1, 3, and 6 monthsmeasured by HPLC. Bars represent mean±s.e.m. One-way ANOVA followed byTukey's post hoc test (n=4 to 6 mice per group). *P<0.05, **P<0.005,***P<0.001.

FIG. 89 shows ipsilateral HVA concentration in the striatum of PBS,α-syn PFF, PAR-α-syn PFF, or PAR-injected mice at 1, 3, and 6 monthsmeasured by HPLC. Bars represent mean±s.e.m. One-way ANOVA followed byTukey's post hoc test (n=4 to 6 mice per group). *P<0.05, **P<0.005,***P<0.001.

FIG. 90 shows contralateral HVA concentration in the striatum of PBS,α-syn PFF, PAR-α-syn PFF, or PAR-injected mice at 1, 3, and 6 monthsmeasured by HPLC. Bars represent mean±s.e.m. One-way ANOVA followed byTukey's post hoc test (n=4 to 6 mice per group). *P<0.05, **P<0.005,***P<0.001.

FIG. 91 shows representative immunoblots of TH, DAT, and β-actin in thestriatum of PBS, α-syn PFF, PAR-α-syn PFF, or PAR-injected mice at 1, 3,and 6 months.

FIG. 92 shows quantification of TH and DAT levels in the striatumnormalized to β-actin. Error bars represent the mean±s.e.m. One-wayANOVA followed by Tukey's post hoc test (n=3-4).

FIG. 93 shows behavioral abnormalities of PBS, α-syn PFF, PAR-α-syn PFF,or PAR-injected mice at 1, 3, and 6 months measured by pole test. Dataare the means s.e.m. One-way ANOVA followed by Tukey's post hoc test.*P<0.05, **P<0.005, ***P<0.001.

FIG. 94 shows behavioral abnormalities of PBS, α-syn PFF, PAR-α-syn PFF,or PAR-injected mice at 1, 3, and 6 months measured by grip strengthtest. Data are the means s.e.m. One-way ANOVA followed by Tukey's posthoc test. *P<0.05, **P<0.005, ***P<0.001.

FIG. 95 shows establishment of PAR ELISA. ELISA detected the PAR as lowas 3 pM and was saturated at 50 nM.

FIG. 96 shows representative immunoblots of PAR, TH, and β-actin in thesubstantia nigra of control and PD patients.

FIG. 97 shows quantification of PAR and TH levels normalized to β-actin.Error bars represent the mean±s.e.m. One-way ANOVA followed by Tukey'spost hoc test (n=5). *P<0.05, ***P<0.001.

DETAILED DESCRIPTION OF THE INVENTION

Previously, it was discovered that a novel and useful mechanism forpreventing cell death following activation of PARP-1 by administeringagents that interfere with the PAR-AIF interaction (see US 20120122765which is incorporated by reference in its entirety). Such mechanisms areuseful in treating patients suffering from Parkinson's disease. In thisregard, the identification of AIF as a PAR polymer-binding proteinestablishes that therapeutic compounds which inhibit the interaction ofPAR polymer with AIF may be useful in mono or combination therapies asprotective compounds against stressors which activate PARP-1.

In continuing this work, disclosed herein is a novel method formonitoring and assessing patients suffering from PD. Also disclosed is anovel method for monitoring and assessing the effectiveness of medicaltreatment administered to a patient suffering from PD. The theranosticmethods disclosed herein enable a medical professional to better developa treatment plan for a patient suffering from PD that has the bestopportunity to slow or arrest the progression of the disease.

In one aspect, disclosed herein is a method for determining thepoly(ADP-ribose) (PAR) concentration in cerebral spinal fluid, themethod comprising: collecting a sample of cerebrospinal fluid (CSF) froma patient, performing a PAR-sandwich ELISA on the CSF sample, therebydetermining the PAR concentration in the CSF.

In some embodiments, the method further comprises comparing the PARconcentration in the CSF sample to the PAR concentration in at least onecontrol sample. The control sample can be a previously collected CSFsample from the same patient or from a different patient. In someaspects, more than one control sample is used. Each control sample canbe from the same patient or a different patient selected independentlyfrom every other control sample.

In some aspects, the control sample is a prepared standard with a knownconcentration of PAR such that it would enable a comparison of thetested sample to the control sample, thereby allowing a determination ofthe PAR concentration in the tested sample. Prepared control standards,such as those shown in FIG. 2, may be used thereby allowing for acomparison of the tested sample to a generated control curve thusenabling quantification of the PAR concentration in the tested sample.

A description of the sandwich-ELISA is provided elsewhere herein. Insome aspects, the capture antibody, the detection antibody, or both areanti-PAR antibodies. They may be monoclonal or polyclonal, and they mayor may not be humanized. In some aspects, the antibody is an anti-PARantibody prepared from a human combinatorial antibody library. In someaspects the human combinatorial library is the HuCAL® Technology fromBio-RAD®. For examples of the HuCAL® Technology, see Knappik, A. et al.,(2000). “Fully synthetic human combinatorial antibody libraries (HuCAL)based on modular consensus frameworks and CDRs randomized withtrinucleotides,” J Mol Biol., 296:57-86, and Prassler, J. et al.,(2011). “HuCAL PLATINUM, a synthetic Fab library optimized for sequencediversity and superior performance in mammalian expression systems,” JMol Biol., 413:261-78, both of which are incorporated by reference fortheir teachings thereof.

The detection antibody is conjugated to at least one agent suitable fordetection by colorimetric or other assay. In some aspects, a seconddetection antibody specific for the first detection antibody is used,and the second detection antibody is conjugated to the agent that issuitable for detection by colorimetric or other assay. In some aspectsthe agent suitable for detection by colorimetric assay is biotin.Horseradish peroxidase (HRP) strongly binds to biotin and reacts with3,3′,5,5′-tetramethylbenzidine (TMB) to form a colored product. In someaspects, the colorimetric assay uses HRP and TMB to measure theconcentration of PAR in a CSF sample of a patient.

In another aspect, disclosed herein is a method for determining thetherapeutic efficacy of a medical treatment for Parkinson's disease, themethod comprising: collecting a sample of cerebrospinal fluid (CSF) froma patient, measuring the poly(ADP-ribose) (PAR) concentration in the CSFsample, and comparing the PAR concentration in the patient to a PARconcentration in at least one control sample.

In some aspects, the control sample is a previously collected sample ofCSF from the same patient. The previously collected sample of CSF mayhave been collected at any time in the past. For example the sample mayhave been collected 1 month, 2 months, 3 months, 4 months, 6 months, 9months, 12 months, 15 months, 18 months, 21 months, 24 months, 27months, 30 months, 33 months or 36 months prior to the currentlycollected sample. In some aspects, the sample is collected at any pointduring the lifetime of the patient. This sample collection can be doneon regular or repeated basis as part of routine monitoring of the healthof a patient. In some aspects, the control sample is taken from ahealthy patient. Healthy as used herein means that the patient is notexhibiting any symptoms of a medical condition that might affect theresults of the test. In some aspects, the healthy patient is notexhibiting any symptoms of PD or similar neurological condition.

It has been previously demonstrated that activation of PARP-1 and theaccumulation of PAR are implicated in the neuropathologies of PD, so insome aspects disclosed herein, an increase in the PAR concentration inthe CSF of a patient indicates that the patient either has PD or is atrisk of developing PD. In some aspects, if the PAR concentration in apatient is higher than the control sample that was previously collectedfrom the same patient, it is indicative that the PD in the patient isworsening. In some aspects, an increase in the PAR concentration in thepatient compared to the control sample that was previously collectedfrom the same patient, it is indicative that the medical treatmentreceived by the patient is not arresting the progression of the disease.

In a patient previously diagnosed with PD, either using the methods andtechniques disclosed herein or by another method, and the patient isreceiving medical treatment for PD, when the PAR concentration in thepatient increases as compared to the PAR concentration of a previouslycollected sample from the same patient, it is indicative that themedical treatment is not arresting the disease. In these instances amedical professional will make a decision as to whether or not tocontinue the same treatment or alter the treatment received by thepatient.

In yet another aspect, disclosed herein is a method of monitoring thedisease progression of a patient with PD, the method comprising:collecting a sample of cerebrospinal fluid (CSF) from the patient,measuring the poly(ADP-ribose) (PAR) concentration in the CSF sample,and comparing the PAR concentration in the patient to a PARconcentration in at least one control sample.

In some aspects, the control sample is a previously collected sample ofCSF from the same patient. The previously collected sample of CSF mayhave been collected at any time in the past. For example the sample mayhave been collected 1 month, 2 months, 3 months, 4 months, 6 months, 9months, 12 months, 15 months, 18 months, 21 months, 24 months, 27months, 30 months, 33 months or 36 months prior to the currentlycollected sample. In some aspects, the sample is collected at any pointduring the lifetime of the patient. This sample collection can be doneon regular or repeated basis as part of routine monitoring of the healthand/or treatment of a patient.

When comparing the PAR concentration to a sample taken from the samepatient at an earlier time period, an increase of the PAR concentrationin the CSF is indicative that the condition of the patient is worsening.If the patient is receiving medical treatment, then an increased PARconcentration would indicate that the treatment is not effective in thepatient. If the PAR concentration is the same or lower, it wouldindicate that the treatment the patient is receiving is effective ineither slowing or arresting the progression of the disease. The PARconcentration is measured using a sandwich-ELISA as disclosed elsewhereherein.

In still yet another aspect, disclosed herein is a method fordiagnosing, or determining that a patient is at risk of developing, PD,the method comprises: collecting a sample of cerebrospinal fluid (CSF)from the patient, measuring the poly(ADP-ribose) (PAR) concentration inthe CSF sample, and comparing the PAR concentration in the patient to aPAR concentration in at least one control sample.

According to this method, in some aspects, a patient is diagnosed withPD, or is at risk of developing PD, if the PAR concentration in the CSFof the patient is greater than a predetermined concentration. In someaspects, that predetermined concentration is the PAR concentration inthe CSF of a healthy patient. In some aspects, measuring the PARconcentration in the CSF of the patient is done using a sandwich-ELISAas disclosed elsewhere herein.

According to this method, if a patient has a PAR concentration higherthan a control sample from either a healthy patient or a control samplepreviously taken from the same patient, then the patient is diagnosedwith PD or is determined to be at risk of developing PD. If the PARconcentration in the CSF of the patient is lower than or equal to thePAR concentration in a negative control or a control sample of a healthypatient, then the patient does not currently have PD or the risk ofdeveloping PD is low at that time. Because the ultimate cause of PD isas yet unknown, it is not possible to conclude that a patient will neverdevelop PD.

The term “theranostics” as used herein refers to the process by which anindividualized treatment plan or therapy is developed for a specificpatient. The word defines the ongoing clinical efforts to develop morespecific, individualized treatments for various diseases, and to combinediagnostic and therapeutic capabilities into a single agent. Therationale arose from the fact that many diseases, such as PD and cancer,are immensely heterogeneous, and current treatments are effective foronly limited patient populations or subpopulations and/or at onlyspecific stages of disease progression. The hope was that a closecombination of diagnosis and therapeutics could provide therapeuticprotocols that are more specific to individuals and, therefore, morelikely to offer improved prognoses.

There are no standard treatments for PD. An individualized treatmentplan is developed for each patient based on their symptoms and overallhealth. Treatment options include medication, surgery, and life stylemodifications. Examples of medicaments used to treat PD include, but arenot limited to, levodopa, dopamine agonists, amantadine,anticholinergics, COMT inhibitors, and MAO-B inhibitors. Surgicaltreatments include, but are not limited to, deep brain stimulation,thalamotomy, pallidotomy, and subthalamotomy. Lifestyle treatmentsinclude, but are not limited to, exercise and diet. Many patients oftenexplore so-called “alternative medicine” which can include herbal andvitamin supplements. Additional treatments are always being studied inclinical trials. In some aspects, a patient is receiving at least onetype of medical treatment specifically for PD. It is not uncommon for apatient to receive multiple different types of medical treatment inorder to determine the optimal combination for the patient. Regardlessof how many different types of medical treatment are being administeredto the patient, the methods and techniques disclosed herein are suitablefor assessing the PAR concentrations in the patient and determining thetherapeutic efficiency of the medical treatment or combination of thosetreatments.

Also disclosed herein is a theranostic method that will aid medicalprofessionals in developing an individualized treatment plan for apatient with PD. The method comprises collecting a sample ofcerebrospinal fluid (CSF) from a patient who is receiving at least onemedical treatment for PD, measuring the poly(ADP-ribose) (PAR)concentration in the CSF sample, and comparing the PAR concentration inthe patient to a PAR concentration in at least one control sample.

The medical treatment may be any treatment disclosed elsewhere herein orit may be any other treatment administered by, or under the supervisionof, a medical professional. In some aspects, if a patient has beenadministered a medicament for at least a time period where themedicament is expected to have an effect, and the PAR concentration inthe patient has increased, then the method further comprises alteringthe manner in which the medicament is administered to the patient.Altering the manner in which the medicament is administered may compriseincreasing or decreasing the dose of the medicament; it may compriseincreasing or decreasing the frequency that the medicament is taken byor administered to the patient; it may include changing or eliminating aspecific medicament being administered to the patient.

In another aspect, if the PAR concentration in the CSF of the patienthas increased compared to a previously collected sample of CSF from thesame patient, the medical professional may alter then medical treatmentreceived by the patient to add in an additional form of treatment. Forexample, if the patient has only received one or more medicaments astreatment, then the medical professional may recommend a surgicalalternative treatment. Alternatively, if the patient has received onemedicament (e.g., levodopa), then the medical professional may recommendthe addition of a second medicament (e.g., a dopamine agonist). Theexact nature in which the medical professional alters the medicaltreatment of the patient will be individualized based on the medicalneeds of the patient and the professional judgment of the medicalprofessional.

As used herein, the term “biomarker” refers to the definitionestablished by the National Institutes of Health Biomarkers DefinitionsWorking Group. It is “a characteristic that is objectively measured andevaluated as an indicator of normal biological processes, pathogenicprocesses, or pharmacologic responses to a therapeutic intervention.”Also disclosed herein is the use of PAR in the CSF of a patient as abiomarker for PD. The methods disclosed elsewhere herein are suitablefor either diagnosing a patient with PD or determining if a patient isat risk for PD. An elevated concentration of PAR in the CSF of a patientis indicative of PD or indicative that a patient is at risk ofdeveloping PD. If a patient is already exhibiting symptoms of PD, thenan elevated concentration of PAR in the CSF of the patient suggests thatthe patient has PD. Symptoms of PD include, but are not limited to,tremors or shaking, changes in writing or speech patterns, loss ofsmell, difficulty sleeping, difficulty walking or moving, slowedmovements, constipation, masked face, dizziness or fainting, or stoopingor hunching over. These early warning signs of PD coupled with anelevated PAR concentration in the CSF of a patient suggest the patienthas PD. Further testing may be necessary to confirm this diagnosis. Alsodisclosed herein is the use of PAR as a biomarker for PD. Detection ofan elevated concentration of PD in the CSF of a patient indicates thatthe patient either has or is at risk of developing PD.

As used herein, “sandwich ELISA,” and illustrated in FIG. 2, is avariant of the traditional ELISA that is highly specific for sampleantigen detection and quantification. The sandwich ELISA quantifiesantigens between two layers of antibodies (i.e., a capture antibody anda detection antibody). The antigen to be measured must contain at leasttwo antigenic epitopes capable of binding to the antibody, since atleast two antibodies act in the sandwich. Either monoclonal orpolyclonal antibodies can be used as the capture and detectionantibodies in a sandwich ELISA system. Monoclonal antibodies recognize asingle epitope that allows for the fine detection and quantification ofsmall differences between antigens while polyclonal antibodies are oftenused as the capture antibody to trap as much of the antigen as possible.One advantage of sandwich ELISA is in the ease of sample preparation,meaning the sample does not have to be purified before analysis. Anotheradvantage of sandwich ELISA is the sensitivity of the technique todetect and quantify specific antigens.

In some aspects of the sandwich ELISA as disclosed herein, the captureantibody is immobilized on a plate, chip or other physical structure. Inthe second step, the immobilized capture antibody is exposed to thesample that includes the target protein. As described herein, the targetprotein is PAR. After a predetermined time period, a first detectionantibody is added to bind to the antigen that is bound to theimmobilized capture antibody. In some embodiments, a second detectionantibody is added to bind to the first detection antibody. In allembodiments, at least one of the detection antibodies will comprise adetectable substrate. The detectable substrate may be an enzyme thatreacts with an additional reagent to form a detectable, andquantifiable, product, or detectable substrate may be detectable withoutfurther reaction. In some embodiments, the detectable substrate isbiotin. In all embodiments, the detectable substrate will be detectableand quantifiable. In some embodiments, the detection of the substratewill be colorimetric.

Human antibodies suitable for use in the sandwich ELISA disclosed hereinare known in the art. For example, human myeloma and mouse-humanheteromyeloma cell lines for the production of human monoclonalantibodies have been described in the art (Kozbor J., Immunol. 133:3001,1984; Brodeur et al., “Monoclonal Antibody Production Techniques andApplications,” Marcel Dekker, Inc., New York, 1987, both of which areincorporated by reference for their teachings thereof). In some aspects,the antibody is an anti-PAR antibody prepared from a human combinatorialantibody library (e.g., HuCAL® Technology from Bio-RAD®).

To determine whether α-syn PFF induces the activation of PARP, levels ofPAR are measured using a highly sensitive and specific PAR monoclonalantibody after administration of α-syn PFF to primary mouse corticalneurons (FIGS. 5 to 12). α-syn PFF (1 μg/ml) induced PARP activationpeaks between 3 to 7 days and remained elevated for up to 14 days (FIG.5). Accompanying the elevation of PAR is the death of neurons asassessed by propidium iodide (PI) staining (FIGS. 6 and 7). Treatment ofcortical neurons with the PARP inhibitors, ABT-888 (veliparib) (10 μM),or AG-014699 (Rucaparib) (1 μM) or BMN 673 (Talazoparib) (10 μM)prevents the α-syn PFF-mediated PARP activation and cell death (FIGS. 6,7, and 8). These PARP inhibitors also reduce α-syn PFF-mediatedphosphorylation of α-syn at serine 129 (p-α-syn) as assessed byimmunostaining (FIGS. 37 and 38), and α-syn aggregation as indicated byimmunoblot analysis (FIGS. 39 and 40), both of which are associated withpathology in α-synucleinopathies. Since PARP-1 plays a major role inparthanatos, PARP-1 was deleted from cortical neurons using CRISPR/Cas9via adeno associated virus (AAV) transduction carrying a guide RNAagainst PARP-1 (FIGS. 9. 10 and 41) or used cortical cultures fromPARP-1 knockouts (FIGS. 11, 12, and 42 to 46). Deletion or knockout ofPARP-1 prevents α-syn PFF-mediated PARP activation and cell death (FIGS.9 to 12, 41 and 42). Knockout of PARP1 also reduces p-α-synimmunostaining and α-syn aggregation as indicated by immunoblot analysis(FIGS. 43 to 46). Treatment of cortical neurons with the broad spectrumcaspase inhibitor Z-VAD-FMK (Z-VAD) partially reduces α-syn PFFtoxicity. The necroptosis inhibitor Necrostatin-1 (Nec-1) and theautophagy inhibitor 3-Methyladenine (3-MA) had no effect, while the PARPinhibitor ABT-888 prevents α-syn PFF toxicity (FIGS. 47 and 48). SincePARP inhibition and knockout of PARP-1 reduces the accumulation ofpathologic α-syn as indicated by a reduction of p-α-syn immunostaining,cell-to-cell transmission of α-syn was assessed as previously described.Knockout of PARP-1 reduces the cell-to-cell transmission of pathologicα-syn (FIGS. 49 to 51). These results taken together suggest that α-synPFF primarily kills neurons through parthanatos and that PARP-1contributes to generation of pathologic α-syn.

Since synthetic α-syn PFF kill primary cortical neurons via parthanatos,experiments were done to determine if parthanatos plays a role in theloss of DA neurons following the intrastriatal injection of α-syn PFFusing a standard and validated protocol (FIGS. 13 to 18). A singleintrastriatal injection of α-syn PFF (5 μg) induces PARP activation asdetermined by assessing PAR levels (FIG. 13). Intrastriatal injection ofα-syn PFF into PARP-1 knockout mice fails to increase PAR levels (FIG.13). A single intrastriatal injection of α-syn PFF leads to anapproximate 50% loss of DA neurons 6 months following the injection inWT mice (FIGS. 14 and 15). In contrast, a single intrastriatal injectionof α-syn PFF into PARP-1 knockout mice fails to induce DA cell loss(FIGS. 14 and 15). WT mice were also fed a diet containing the PARPinhibitor ABT-888 (125 mg/kg) and compared with mice given a controldiet (FIGS. 14 and 15). Mice treated with ABT-888 exhibit significantlyless loss of DA neurons after an intrastriatal injection of α-syn PFFcompared to mice on the control diet (FIGS. 14 and 15). ABT-888 alsoreduces the formation of α-syn PFF induced increase in PAR levels (FIG.52). Tyrosine hydroxylase (TH) and dopamine transporter (DAT) levels arealso reduced in WT mice in response to α-syn PFF as determined bywestern blot analysis, while the reduction in TH and DAT levels isprevented in PARP-1 knockout more or ABT-888 treated ST mice (FIGS. 52to 54). Accompanying the loss of DA neurons is a reduction in striatalDA and its metabolites as determined by HPLC in WT mice, but not PARP-1knockout mice or ABT-888 treated mice (FIGS. 16, 57, 58 and 59). Aninjection of intrastriatal α-syn PFF leads to α-syn pathology asassessed by western blotting (FIGS. 52, 55 and 56) and immunostainingfor p-α-syn in DA neurons of WT mice (FIGS. 60 to 63). α-syn pathologyis markedly reduced in PARP-1 knockout mice and ABT-888 treated WT miceconsistent with the absence and reduction of neurodegeneration,respectively. Intrastriatal injection of α-syn PFF causes deficits onthe pole test, a sensitive behavioral measurement of dopaminergicfunction in WT mice, whereas there are no deficits in PARP-1 knockoutmice and ABT-888 WT treated mice (FIGS. 17 and 64). Both forelimb plushindlimb and forelimb grip strength are also reduced in WT mice afterα-syn PFF injection, but not in PARP-1 knockout or ABT-888 treated WTmice (FIGS. 18 and 65). Taken together, these results indicate that thestriatal α-syn PFF-induced loss of DA neurons is dependent on PARP-1.

Since PAR causes liquid demixing of intrinsically disordered proteinsleading to their aggregation, experiments were performed to determinewhether PAR could seed and accelerate α-syn aggregation. Recombinantα-syn was incubated at 37° C. and agitated in the presence and absenceof 5 nM PAR, concentrations that are observed in brain tissue. Highmolecular weight forms of α-syn in the absence of PAR are observed asearly as 4 hours of incubation and α-syn continues to fibrillize withtime (FIG. 19). Different molecular weight forms of α-syn are observedat 72 hours. In the presence of PAR, the fibrillization of α-syn ismarkedly accelerated with different molecular weight forms of α-synbeing observed as early as 24 h of incubation (FIG. 19). Thioflavin Tfluorescence also indicates that PAR accelerates the fibrillization ofα-syn, while PAR alone has no effect on thioflavin T fluorescence (FIG.20). The effect of temperature on PAR mediated acceleration of thefibrillization of α-syn was also assessed (FIG. 66). PAR causes thefibrillization of α-syn at temperatures that are not permissive forα-syn fibrillization in the absence of PAR (FIG. 66). Transmissionelectron microscopy (TEM) was used to monitor the formation of α-synfibrils. At 12 hours α-syn fibrils begin to form in the absence of PARand become fully formed and extensive after 72 hours of agitation andincubation at 37° C. (FIG. 3C). In contrast, in the presence of PAR,α-syn is extensively fibrillated at 12 hours and becomes moreextensively fibrillated at 24 and 72 hours (FIG. 21). At 36 hours theconcentration dependence of α-syn fibrillization in the presence of PARwas monitored. 1 nM PAR is capable of enhancing α-syn fibrillizationwith peak aggregation occurring at 5 nM PAR while 20 nM PAR does notappreciably increase α-syn fibrillization (FIGS. 67 and 68). Since PARis a highly negatively charged molecule, the effects of another highlycharged polymer were tested, PolyA, and which had no effects on α-synfibrillization (FIGS. 69 and 70). ADP ribose monomer also had no effecton α-syn fibrillization (FIGS. 69 and 70). To determine whetherendogenous PAR formation can accelerate α-syn fibrillization, primarymouse cortical neurons overexpressing WT human α-syn following AAV-α-syntransduction were treated with a toxic dose of N-methyl-D-aspartate(NMDA). In WT cultures NMDA treatment leads to activation of PARP asassessed by PAR immunoblot and a concomitant aggregation of α-syn, whileα-syn did not aggregate in PARP-1 KO cultures treated with NMDA (FIG.22). Exogenous administration of PAR via Bioporter increases theaggregation of α-syn in both WT and PARP-1 KO cultures transduced withAAV-α-syn (FIG. 71), indicating that PAR, not PARP-1, can directlyincrease α-syn aggregation. Two different PARP inhibitors, ABT888 andAG014699, prevent α-syn aggregation and PARP activation in response toNMDA administration (FIG. 23). In SH-SY5Y cells the potent PARPactivator N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) enhances theaggregation of overexpressed WT or A53T α-syn in SH-SY5Y cells, whileMNNG has no effect in PARP-1 KO SH-SY5Y cells (FIGS. 72 and 73).Exogenous administration of PAR increased the aggregation of α-syn inboth SH-SY5Y WT and SH-SY5Y PARP-1 KO cultures (FIG. 74). Two differentPARP inhibitors, ABT888 and AG014699, prevent α-syn aggregation and PARPactivation in response to MNNG administration in SH-SY5Y cells (FIG.75).

To determine whether PAR changes the biophysical properties of α-synPFF, a series of biochemical analysis were conducted using α-syn PFF andα-syn PFF in the presence of PAR (PAR-α-syn PFF). First, proteinase K(PK) digestion of α-syn PFF was performed and monitored by α-synimmunoblots. α-syn PFF and PAR-α-syn PFF showed very distinct bandingpatterns after PK digestion, with PAR-α-syn PFF's being more resistantto increasing concentrations of PK (FIG. 24). PAR-α-syn PFF showedpredominantly an undigested band of α-syn (1^(st) band) with comparabledigested bands only at higher concentration of PK, while α-syn PFFdegraded into smaller fragments (2^(nd) to 5^(th) band) at lowerconcentration of PK (0.5 and 1 μg/ml) and these bands became predominantat higher concentration of PK (1.5-2.5 μg/ml) (FIG. 24). Using epitopespecific antibodies to α-syn, it was found that PAR renders the majorityof the α-syn regions resistant to PK digestion (FIG. 76). The resistanceto PK digestion of PAR-α-syn PFF suggests that PAR induces the formationof distinct α-syn PFF strains with more misfolded and compact structurethan α-syn PFF. α-syn PFF and PAR-α-syn PFF-induced neuronal cell deathwere then compared in cultured neurons. After 14 days of treatment,there is enhanced cell death in cultures treated with PAR-α-syn PFF ascompared to that with α-syn PFF (FIG. 77). PAR itself does not causesignificant cell death even at higher concentration (20 nM) (FIG. 77).To further confirm the potencies of neuropathology, p-α-synimmunoreactivity was monitored after treatment of varying concentrationof α-syn PFF or PAR-α-syn PFF. P-α-syn immunoreactivity was observed at20 ng of PAR-α-syn PFF at an equivalent level to 500 ng of α-syn PFF,suggesting the PAR modification of α-syn PFF increases toxicity by 25fold (FIGS. 78 and 79). PAR-α-syn PFF significantly increased p-α-synimmunoreactivity at higher concentrations (FIGS. 78 and 79).Phospho-α-syn immunoreactivity at different time points was alsomonitored in cultured neurons exposed to α-syn PFF or PAR-α-syn PFF. Inthe absence of PAR, α-syn PFF treatment leads to barely detectablep-α-syn immunoreactivity 1 day post treatment, while PAR-α-syn PFFtreatment leads to detectable levels of p-α-syn immunoreactivity asearly as 1 day post treatment and markedly enhances immunoreactivity at7 days (FIG. 25). Immunoblot analysis of α-syn indicates that aggregatedand phosphorylated α-syn are detectable at 4 days, while in the absenceof PAR these species of α-syn are only detectable after 7 days oftreatment. In the presence of PAR there is an increase in the aggregatedform of α-syn (FIG. 26).

To determine whether the PAR-α-syn PFF strain exhibits enhancedneurotoxicity in vivo, a single intrastriatal injection of PAR-α-syn PFF(5 μg) was compared to that of α-syn PFF (5 μg). There is trend towardthe loss of DA neurons ipsilateral to the injection side of SNpc afterone month and a significant loss of DA neurons after 3 months followingPAR-α-syn PFF injection, while α-syn PFF injection has no effect atthese time points (FIGS. 27, 28 and 80). Six months after PAR-α-syn PFFor α-syn PFF injection there is no significant difference in the loss ofDA neurons (FIGS. 27, 28 and 80). There is no significant loss of DAneuron contralateral to the injection side at any time point (FIGS. 81and 82). PAR injection by itself has no effect on DA neuron number(FIGS. 27, 28, and 80). PAR-α-syn PFF also accelerate the loss ofstriatal DA and its metabolites as determined by HPLC with significantreductions in DA and its metabolites being observed 1 month after thePAR-α-syn PFF injection in contrast to α-syn PFF (FIGS. 29 and 83 to90). Three and 6 months after PAR-α-syn PFF or α-syn PFF injection thereis no significant difference in the loss of DA and its metabolites(FIGS. 29 and 83 to 90). TH and DAT levels are also reduced afterPAR-α-syn PFF compared to α-syn PFF as determined by western blotanalysis three months after the injection, while there is no differencein the degree of loss at 6 months (FIGS. 91 to 94). α-syn pathology asassessed by immunostaining for p-α-syn in DA neurons was increased byPAR-α-syn PFF compared to α-syn PFF at 3 months and 6 months afterinjection (FIGS. 30 and 31). PAR-α-syn PFF caused a deficit on the poletest at 3 months consistent with loss of DA neurons and DA deficits at 3months, whereas there are no significant deficits in α-syn PFF or PARinjected mice (FIGS. 32 and 93). Both forelimb plus hindlimb andforelimb grip strength were also reduced in PAR-α-syn PFF, but not α-synPFF or PAR inject mice at 3 months (FIGS. 33 and 94). At 6 months thereis no significant difference in the behavioral deficits induced byPAR-α-syn PFF or α-syn PFF (FIGS. 32, 33, 92 and 94). Taken together,these results indicate that PAR-α-syn PFF are substantially moreneurotoxic then α-syn PFF in vivo.

To determine whether PAR plays a role in patients with PD, PAR levelswere monitored in the CSF of patients with PD versus controls (Table 1)using a sensitive ELISA for PAR (FIG. 95). PAR levels are elevated in PDpatients compared to controls in two independent patient cohorts (FIGS.34 and 35). As previously reported, PAR levels are increased in thesubstantia nigra of patients with PD compared to controls (FIGS. 96 and97, and Table 2). PAR immunoreactivity is colocalized with α-syn in LewyBodies of PD patients (FIG. 36).

The results indicate that α-syn PFF kills neurons both in vitro and invivo via activation of PARP-1 in a cell death process designatedparthanatos. Knockout of PARP-1 and inhibition of PARP prevents theneurodegeneration and behavioral deficits initiated by an intrastriatalα-syn PFF injection. Activation of parthanatos seems to be the primarydriver of α-syn PFF neurodegeneration since necroptosis and autophagyinhibitors have no effect on α-syn PFF neurotoxicity and there is onlymodest protection by caspase inhibition. It is known that α-syn PFFinduce inflammatory mediator activation, which likely contributes, inpart, to cell death and accounts for the modest neuroprotection by thebroad spectrum caspase inhibitor, ZVAD.

Recent studies have identified conformational variants of α-syn strainsthat exhibit distinct neurotoxicity, seeding abilities and propagation,which contribute to different properties of α-synucleinopathies. Giventhat α-syn PFF induces PARP activation and PAR accumulation, PAR thenaccelerates α-syn fibrillization and changes the biochemical propertiesof α-syn PFF converting it to a more toxic strain. Consistent with thisnotion are observations that PAR-α-syn PFF shows an approximate 25 foldincrease in α-syn aggregation and neurotoxicity compared to the parentalα-syn PFF. Moreover PAR-α-syn PFF-injected mice show an accelerateddisease progression and phenotype compared to α-syn PFF injected mice.In addition to PAR levels being increased in cultured neurons and mousebrain, it was observed that PAR levels in PD are elevated not only inthe substantia nigra, but also the CSF. The elevation of PAR in the CSFand brains of PD patients and evidence of PARP activation in thesubstantia nigra of PD patients suggests that PARP activationcontributes to the pathogenesis of PD through parthanatos and conversionof α-syn to a more toxic strain.

This written description uses examples to disclose the invention,including the best mode, and also to enable any person skilled in theart to practice the invention, including making and using any devices orsystems and performing any incorporated methods. The patentable scope ofthe invention is defined by the claims, and may include other examplesthat occur to those skilled in the art. Such other examples are intendedto be within the scope of the claims if they have structural elementsthat do not differ from the literal language of the claims, or if theyinclude equivalent structural elements with insubstantial differencesfrom the literal languages of the claims.

EXAMPLES

Animals

C57BL/6 mice were obtained from the Jackson Laboratories (Bar Harbor,Me.). PARP-1 KO mice were obtained from The Jackson Laboratory (BarHarbor, Me., USA). The littermates of WT and PARP1 KO mice were used inexperiments. All housing, breeding, and procedures were performedaccording to the NIH Guide for the Care and Use of Experimental Animalsand approved by Johns Hopkins University Animal Care and Use Committee.

Preparation of α-syn PFF and PAR-α-syn PFF

Recombinant mouse α-syn proteins were purified. α-syn PFF were preparedin PBS by constantly agitating α-syn with a thermomixer (1,000 rpm at37° C.) (Eppendorf). After 7 days of incubation, the α-syn aggregateswere diluted to 0.1 mg/ml with PBS and sonicated for 30 s (0.5 sec pulseon/off) at 10% amplitude (Branson Digital Sonifier, Danbury, Conn.,USA). The α-syn PFF were kept at −80° C. until use. Synthesis andpurification of PAR polymer were performed as described elsewhere.PAR-α-syn PFF was prepared by adding 5 nM or indicated dose of PAR inα-syn fibrillization reaction.

Stereotaxic Injection of α-Syn PFF

Two to 3-month-old WT and PARP1 KO mice were deeply anesthetized with amixture of ketamine (100 mg/kg) and xylazine (10 mg/kg). PBS, α-syn PFF,PAR-α-syn PFF or PAR was unilaterally injected into striatum (2 μl perhemisphere at 0.4 μl/min) with the following coordinates:anteroposterior (AP)=+2.0 mm, mediolateral (ML)=±2.0 mm, dorsoventral(DV)=+2.8 mm from bregma. After the injection, the needle was maintainedfor an additional 5 min for a complete absorption of the solution. Aftersurgery, animals were monitored and post-surgical care was provided.Behavioral tests were performed 1, 3 and 6 months after injection andmice were euthanized for biochemical and histological analysis. Forbiochemical studies, tissues were immediately dissected and frozen at−80° C. For histological studies, mice were perfused with PBS and 4% PFAand brains were removed, followed by fixation in 4% PFA overnight andtransfer to 30% sucrose for cryoprotection.

Thioflavin T (ThT) Binding Assay

α-syn fibrillization with or without PAR was monitored with ThTfluorescence. Aliquots of 5 μL from the incubation mixture were taken atvarious time points, diluted to 100 μL with 25 μM ThT in PBS, andincubate for 10 min at room temperature. The fluorescence was recordedat 450 nm excitation and 510 nm emission using SpectraMax plate reader(Molecular Devices, Sunnyvale, Calif.). The experiments were performedin triplicate.

Transmission Electron Microscopy (TEM) Measurements

α-syn PFF or PAR-α-syn PFF were adsorbed to glow discharged 400 meshedcarbon coated copper grids (Electron Microscopy Sciences) for 2 min,quickly washed twice with Tris-HCl (50 mM, pH 7.4), and floated upon twodrops of 0.75% uranyl formate for 30 s each. The grids were allowed todry before imaging on a Phillips CM 120 TEM operating at 80 kV. Theimages were captured and digitized with an ER-80 CCD (8 megapixel) byadvanced microscopy techniques.

Intracellular Delivery of PAR

Purified PAR was intracellularly delivered using BioPORTER (Genelnatis,San Diego, Calif.) according to the manufacturer's instructions. PARpolymer was diluted to desired concentration with PBS. The dilutedsolution was added to the dried BioPORTER reagent and mixed gently,followed by incubation at room temperature for 5 min. The BioPORTER-PARcomplex was added to cell culture after a wash in serum-free media andincubated for 3-4 h at 37° C. Cultures were subsequently used forexperiments.

Tissue Lysate Preparation and Western Blot Analysis

Human post mortem brain (Table 1) or mouse brain tissue were homogenizedand prepared in lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mMEDTA, 1% Triton x-100, 0.5% SDS, 0.5% sodium-deoxycholate, phosphataseinhibitor mixture I and II (Sigma-Aldrich, St. Louis, Mo., USA), andcomplete protease inhibitor mixture (Roche, Indianapolis, Ind., USA)],using a Diax 900 homogenizer (Sigma-Aldrich). After homogenization,samples were rotated at 4° C. for 30 min for complete lysis, thehomogenate was centrifuged at 15,000 g for 20 min and the supernatantswere used for further analysis. Protein levels were quantified using theBCA assay (Pierce, Rockford, Ill., USA), samples were separated usingSDS-polyacrylamide gels and transferred onto nitrocellulose membranes.The membranes were blocked with 5% non-fat milk in TBS-T (Tris-bufferedsaline with 0.1% Tween-20) for 1 h, probed using primary antibodies(Table 3) and incubated with appropriate HRP-conjugated secondaryantibodies. The bands were visualized by ECL substrate.

TABLE 1 Clinical Information of Control and PD CSF used in FIG. 34 GroupAge Sex Control 72 F (n = 31) 82 F 82 M 56 F 70 F 69 F 67 F 57 F 70 F 66F 78 M 46 F 61 F 68 M 54 F 66 F 46 F 76 M 68 M 74 M 57 F 64 F 65 F 69 F82 F 71 M 55 M 67 F 60 M 74 F 69 M PD 66 M (n = 80) 70 M 87 M 56 F 69 F55 F 72 M 66 M 56 F 78 M 70 F 59 F 57 M 61 M 61 M 73 M 71 M 57 M 69 F 62M 51 F 68 F 59 F 61 M 70 M 64 F 74 M 63 M 70 M 67 M 67 M 74 F 67 M 57 M72 M 56 F 77 F 68 M 75 F 67 M 52 M 55 M 75 M 67 M 63 M 65 F 74 F 69 M 76M 64 M 56 M 60 F 58 F 49 M 49 M 66 M 54 M 74 F 64 M 64 M 68 M 68 F 67 F68 M 70 M 63 M 64 M 78 M 69 M 58 F 61 M 68 M 79 M 66 M 56 F 66 M 68 M 74M 75 M 64 F Summarized information Group Mean age ± s.e.m. F/M ratio (%)Control 66.9 ± 1.69 67.7/32.3 PD 66.0 ± 0.85 31.2/68.8

Cell Culture, Transfection, Primary Neuronal Culture and Treatment

SH-SY5Y cells (ATCC) were cultured in DMEM containing 10% fetal bovineserum and penicillin/streptomycin at 37° C. under 5% CO₂. The cells weretransfected using PolyFect reagent (Qiagen) according to themanufacturer's instructions. Primary cortical neurons from WT or PARP1KO embryos were prepared. Briefly, the primary cortical neurons werecultured at embryonic day 16 in neurobasal media supplemented with B-27,0.5 mM L-glutamine, penicillin and streptomycin (Invitrogen, GrandIsland, N.Y.). The neurons were replaced to fresh medium every 3-4 days.ABT-888 (10 μM), AG-014699 (1 μM), BMN 673 (10 μM), Z-VAD (20 μM), Nec-1(20 μM) or 3-MA (500 μM) was pretreated to neurons 1 h before α-syn PFFtreatment. α-syn PFF were added at 7 days in vitro (DIV) and furtherincubated for indicated times followed by cell death assay orbiochemical experiments. Primary neurons were infected with AAV2-controlsgRNA or AAV2-PARP1 sgRNA (ViGene Bioscineces, Rockville, Md., USA), andAAV-α-syn WT or AAV-α-syn A53T at DIV 4-5.

Cell Death Assessment

Primary cultured cortical neurons were treated with α-syn PFF orPAR-α-syn PFF for 14 days. Percent of cell death was determined bystaining with 7 μM Hoechst 33342 and 2 μM propidium iodide (PI)(Invitrogen, Carlsbas, Calif.). Images were taken and counted by Zeissmicroscope equipped with automated computer assisted software(Axiovision 4.6, Carl Zeiss, Dublin, Calif., USA).

Microfluidic Chambers

Triple compartment microfluidic devices (TCND1000) were obtained fromXona Microfluidic, LLC (Temecula, Calif., USA). Glass coverslips wereprepared and coated before being affixed to the microfluidic device.Approximately 100,000 WT or PARP1 KO neurons were plated per chamberindividually. At 7 DIV, 0.5 mg α-syn PFF were added into chamber 1. Tocontrol for direction of flow, a 50 μl difference in media volume wasmaintained between chamber 1 and chamber 2 and chamber 2 and chamber 3.Neurons were fixed on day 14 after α-syn PFF treatment using 4%paraformaldehyde in PBS. The chambers were then processed forimmunofluorescence staining with p-α-syn antibody.

Behavioral Tests

The behavioral deficits in α-syn PFF injected WT or PARP-1 KO mice,α-syn PFF injected mice fed ABT-888, and α-syn PFF or PAR-α-syn PFFinjected mice were assessed by the pole test and the grip strength test1 week prior to sacrifice. All the experiments were performed in a blindfashion.

Pole Test.

A metal rod (75 cm long with a 9 mm diameter) wrapped with bandage gauzewas used as the pole. Before the actual test, the mice were trained fortwo consecutive days and each training session consisted of three testtrials. Mice were placed on 7.5 cm from the top of the pole and the timeto turn and total time to reach the base of the pole were recorded. Theend of test was defined as placing all 4 paws on the base. The maximumcutoff time to stop the test and recording was 60 s. After each trial,the maze was cleaned with 70% ethanol.

Grip Strength Test.

Neuromuscular function was measured by determining the maximal peakforce developed by the mice using an apparatus (Bioseb, USA). Mice wereplaced onto a metal grid to grasp with either fore or both limbs thatare recorded as ‘fore limb’ and ‘fore and hindlimb’, respectively. Thetail was gently pulled and the force applied to the grid before the micelose grip was recorded as the peak tension displayed in grams (g).

Dopamine and Derivatives Measurement Using HPLC

Biogenic amine concentrations were measured by high-performance liquidchromatography with electrochemical detection (HPLC-ECD). The striatumwas rapidly removed from the brain, weighted and sonicated in ice cold0.01 mM of perchloric acid containing 0.01% EDTA. The 60 ng of3,4-dihydroxybenzylamine (DHBA) was used as an internal standard. Aftercentrifugation at 15,000 g for 30 min at 4° C., the supernatant wascleaned using a 0.2 μm filter and 20 μM of supernatant was analyzed inthe HPLC column (3 mm×150 mm, C-18 reverse phase column, Acclaim™ PolarAdvantage II, Thermo Scientific, USA) by a dual channel coulochem IIIelectrochemical detector (Model 5300, ESA, Inc. Chelmsford, Mass., USA).The protein concentrations of tissue homogenates were measured using theBCA protein assay kit (Pierce, Rockford, Ill., USA). Data werenormalized to protein concentrations and expressed in ng/mg protein.

Immunohistochemistry and Immunofluorescence

Mice were perfused with PBS and 4% PFA and brains were removed, followedby fixation in 4% PFA overnight and transfer to 30% sucrose forcryoprotection. Immunohistochemistry (IHC) and immunofluorescence (IF)was performed on 40 μm thick serial brain sections. Primary antibodiesand working dilutions are detailed in Table 2. For histological studies,Free-floating sections were blocked with 10% goat serum in PBS with 0.2%Triton X-100 and incubated with TH or p-α-syn antibodies followed byincubation with biotin-conjugated anti-rabbit or mouse antibody,respectively. After three times of washing, ABC reagent (Vectorlaboratories, CA, USA) was added and the sections were developed usingSigmaFast DAB peroxidase substrate (Sigma-Aldrich). Sections werecounterstained with Nissl (0.09% thionin). For the quantification, bothTH- and Nissl-positive DA neurons from the SNpc region were countedthrough optical fractionators, the unbiased method for cell counting,using a computer-assisted image analysis system consisting of anAxiophot photomicroscope (Carl Zeiss) equipped with a computercontrolled motorized stage (Ludl Electronics, Hawthorne, N.Y., USA), aHitachi HV C20 camera, and Stereo Investigator software(MicroBright-Field, Williston, Vt., USA). The total number of TH-stainedneurons and Nissl counts were analyzed. For immunofluorescent studies,double-labeled sections with TH and p-α-syn antibodies were incubatedwith a mixture of Alexa-fluor 488- and 594-conjugated secondaryantibodies (Invitrogen, Carlsbad, Calif., USA). The fluorescent imageswere acquired by confocal scanning microscopy (LSM710, Carl Zeiss). Allthe images were processed by the Zen software (Carl Zeiss). The selectedarea in the signal intensity range of the threshold was measured usingImageJ software.

TABLE 2 Clinical Information of Control and PD CSF used in FIG. 35 GroupAge Sex Control 71 M (n = 33) 62 F 63 M 63 F 74 F 65 M 62 M 64 M 79 M 66M 27 M 60 F 55 M 60 M 62 F 60 F 65 F 66 F 27 M 63 F 62 M 62 F 60 F 74 M61 M 66 M 76 F 65 F 61 F 71 M 71 F 63 F 74 M PD 50 M (n = 21) 67 F 76 M57 F 54 M 51 F 56 F 50 M 66 M 53 M 56 F 50 M 43 M 75 M 58 F 69 M 62 F 62M 85 M 73 F 82 F Summarized information Group Mean age ± s.e.m. F/Mratio (%) Control 62.76 ± 1.83 48.5/51.5 PD 61.67 ± 2.52 42.9/57.1

PK Digestion of α-Syn PFF

PK digestion was performed. Ten micrograms of α-syn PFF or PAR-α-syn PFFwere mixed with 0.5 to 2.5 ug/ml of PK in PBS and incubated at 37° C.for 30 min. The reaction was stopped by adding 1 mM PMSF, boiled withSDS-sample buffer for 5 min. The bands of the PK digestion products weredetected by immunoblotting using epitope-specific α-syn antibodies(Table 3).

TABLE 3 List of Some of the Antibodies Tested in this Study AntibodySource Identifier Dilution PAR Made in N/A 1:2,000 (WB) Dawson lab 1:500(IF) PARP-1 BD Bioscience 611039 1:2,000 (WB for human) Cell Signaling 9532 1:1,000 (WB for mouse) α-syn BD Bioscience 610787 1:3,000 (WB)1:500 (IHC) α-syn (121-125), Sigma S5566 1:3,000 (WB) Syn211 α-syn(N-term) LSBio LS- 1:3,000 (WB) C352877 α-syn (115-122), LB509 Abcamab27766 1:3,000 (WB) α-syn (61-95), 5C2 Novus NBP1- 1:3,000 (WB) 04321p-α-syn (Ser129) Biolegend 825701 1:1,000 (WB) 1:500 (IF) TH NovusNB300-19 1:2,000 (WB) Biologicals 1:1,000 (IHC, IF) DAT Sigma D69441:1,000 (WB) GAPDH Abcam ab8245 1:5,000 (WB) β-actin-HRP Sigma A38541:20,000 (WB)

Human Clinical Trials

Human CSF Samples and PAR ELISA

Participants at the Johns Hopkins University site of the NINDSParkinson's Disease Biomarker Program (PDBP) underwent extensiveclinical and cognitive testing and a lumbar puncture annually. The CSFwas centrifuged, aliquoted, and stored at −80° C. within one hour ofacquisition. Two different clones (#19 and #25) of monoclonal anti-PARantibody were used for PAR ELISA. Anti-PAR antibody (capture antibody,clone #19) (5 ug/ml) was coated on 96-well microtiter plate (NUNC, Cat#46051), various concentration of purified PAR (0-200 nM, positivecontrol) and CSF samples from either normal or PD patients were added toeach well and incubated for 1 h at room temperature (RT). After washingthe plates five times with PBST (0.05% Tween20 in PBS buffer), thebiotinylated PAR antibody (detection antibody, clone #25) was incubatedfor 1 h at RT. The color change was detected via HRP-conjugatedstreptavidin antibody (Thermo Scientific). The assay can detect the PARas low as 3 pM and is saturated at 50 nM.

Clinical Dementia Rating

The Clinical Dementia Rating (CDR) scale is a five-point scale used toassess six different areas of cognitive and functional performanceapplicable to patients with neurological degeneration and dementia. SeeHughes C P, Berg L, Danziger W L, Coben L A, Martin R L, “A New ClinicalScale for the Staging of Dementia,” Br J Psychiatry, (1982) 140:566-72which is incorporated by reference for its teaching thereof. The sixareas are memory, orientation, judgment & problem solving, communityaffairs, home & hobbies, and personal care. All patients were assessedusing the CDR scale and a personal interview to determine if they hadnormal cognition, mild cognitive impairment, or dementia.

Student's t-test were used to compare the concentration of PAR betweencontrols and individuals with PD and those with normal cognition andcognitive impairment. A generalized linear model then evaluated thedeterminants of change in PAR concentration and whether PARconcentration was associated with cognitive changes.

Human Combinatorial Antibody Library

The HuCAL® technology presents an alternative method to the conventionalmethods of obtaining custom antibodies. Whereas the production ofmonoclonal antibodies requires immunizing a mouse, rabbit, or goat, andsubsequently extracting the B cells from the spleen to recover theantibodies presented, HuCAL® technology allows for faster productiontime. Through the use of library of complementarity determining regions(CDR), 6 light chain variable regions (V_(L)) and 7 heavy chains(V_(H)), it is possible to generate billions of antibodies in vitro.This is paired with a phage display that incorporates the antibody genesinto bacteriophages that presents the antibody on the coating via adisulfide bond, thereby presenting a physical linkage of the phenotypeand genotype. Reductive cleavage of the disulfide bond allows for therecovery of the antibodies following a screening, independent of theaffinity to the antigen. AbD Serotec was provided with syntheticpurified terminally-biotinylated PAR polymer. It was synthesized throughreductive amination of pure PAR polymer (2-300 mer). The antibody cloneswere selected for binding to only polymers and oligomers, and not theADP-ribose monomer.

The HuCAL® antibody generation process begins with the immobilization ofthe antigen (PAR) using a covalent coupling to magnetic beads. These aresubsequently incubated with the HuCAL® library where nonspecificantibodies are washed out and the specific antibody-phages are eluted.E. coli cultures are subjected to infection from the specificantibody-phages to generate an enriched antibody library for thesubsequent round of phage screening. The DNA from these enrichedantibody-phases is retrieved and subcloned into Fab expression vectorsand plated into E. coli colonies to produce the Fab fragments. Followingthis are the colony picking, primary screening, where the colonies weregrown in a 384-microtiter plate. Antibody expression is induced andcollected after lysing the cultures. These cultures are screened byELISA with the terminally labelled-PAR antigen. The positive hits fromthe primary screening are then sequenced to identify the uniqueantibodies, which are stored for future synthesis for reproducibility.Secondary screening was performed to select out PAR monomer bindingantibodies. Finally, expression and purification through affinitychromatography was performed to obtain antibody clones.

Sandwich-ELISA

Anti-PAR antibody (capture antibody, clone #19) (5 μg/ml) was coated ona 96-well microtiter plate (NUNC, Cat #46051). Different concentrationsof purified PAR (0-200 nM, positive control) and CSF samples from eithernormal or PD patients were added to each well, and incubated for 1 hr atroom temperature (RT). After washing the plates five times with PBST(0.05% Tween20 in PBS buffer), the biotinylated PAR antibody (detectionantibody, clone #25) was incubated for 1 hr at RT. The color change wasdetected via HRP-conjugated streptavidin antibody. This assay detectsthe PAR concentration as low as 3 pM and is saturated at 50 nM.

Results

One hundred ten individuals contributed CSF at baseline (80 PD, 30control), 94 individuals contributed CSF at the first follow-up (68 PD,26 controls), 71 individuals contributed CSF at the second follow-up (51PD, 20 controls), and 36 individuals contributed CSF at the thirdfollow-up (28 PD, 8 controls). At baseline, the average age for both PDand controls was approximately 66 years (p=0.71) and 67% of PD patientswere men while 37% of controls were men (p<0.01). Mean PD duration was6.7 years. There were differences in the mean concentration of PARbetween individuals with PD and controls at the first three visits, witha trend toward a difference in the 4th visit (visit 1: PD mean 112.13,control mean 87.99 p=0.04; visit 2: PD mean 145.49, control mean 110.63p=0.04; visit 3: PD mean 132.29, control mean 86.06 p=0.01; visit 4: PDmean 151.88, control mean 111.07 p=0.08).

PAR Concentration (pM) PD Patients Healthy Controls Visit 1 112.13 87.99Visit 2 145.49 110.63 Visit 3 132.29 86.06 Visit 4 151.88 111.07

Disease status was a significant predictors of PAR concentration(p<0.01), even after controlling for age, gender, MDS-UPDRS Motorscores, levodopa equivalent dosing, and cognitive impairment (see FIGS.3 and 4). PAR concentration at visit 2 and visit 4 were significantlydifferent from PAR concentration at visit 1 (p<0.01, p=0.01) Among onlyPD participants, PAR concentration (p=0.03) and MDS-UPDRS Motor scores(p<0.01) were significant predictors of cognitive decline.

Statistical Analysis

All data are represented as mean±s.e.m. with at least 3 independentexperiments. Statistical analysis was performed using GraphPad Prism.Differences between 2 means and among multiple means were assessed byunpaired two-tailed student t test and ANOVA followed by Tukey's posthoc test, respectively. Significance was assessed as *P<0.05, **P<0.005,and ***P<0.001.

In summary, there was a significant difference in the PAR concentrationbetween PD and control patients. There was also a significant differencein the PAR concentration between patients on subsequent samplecollection visits.

1. A method for determining a poly(ADP-ribose) (PAR) concentration incerebral spinal fluid (CSF), said method comprising collecting a sampleof cerebrospinal fluid from a patient, performing a PAR-sandwich ELISAon said CSF sample, thereby determining the PAR concentration in theCSF.
 2. The method according to claim 1, further comprising comparingthe PAR concentration in said CSF sample to at least one control sample.3. The method according to claim 1, wherein the sandwich ELISA comprisesat least at capture antibody and a detection antibody, and said captureantibody, said detection antibody or both are an anti-PAR monoclonalantibody.
 4. The method according to claim 3, wherein the captureantibody is attached to a solid support.
 5. The method according toclaim 3, wherein the anti-PAR monoclonal antibody is fully human.
 6. Themethod according to claim 1, wherein comparing the PAR concentration inthe CSF sample to at least one control sample is done by colorimetricassay.
 7. The method according to claim 3, wherein the detectionantibody is conjugated to biotin.
 8. A method for determining thetherapeutic efficacy of a medical treatment for Parkinson's disease,said method comprising collecting a sample of cerebrospinal fluid (CSF)from a patient receiving at least one medical treatment for PD,measuring a poly(ADP-ribose) (PAR) concentration in said CSF sample, andcomparing the PAR concentration in said patient to a PAR concentrationin at least one control sample.
 9. The method according to claim 8,wherein the control sample is a CSF sample collected from said patientat an earlier time period.
 10. The method according to claim 8, whereinthe control sample is a CSF sample collected from a healthy donor. 11.The method according to claim 9, wherein if the PAR concentration insaid patient is higher than the PAR concentration in the control sample,then the medical treatment is not effective for said patient, and if thePAR concentration in said patient is the same or lower than the PARconcentration in the control sample, then the medical treatment iseffective for said patient.
 12. The method according to claim 8, whereinthe medical treatment is administering to said patient one or moremedicaments, performing surgery on said patient or a combinationthereof.
 13. The method according to claim 8, wherein measuring the PARconcentration in said CSF sample is by sandwich ELISA.
 14. A method formonitoring the disease progression of a patient with Parkinson's disease(PD), said method comprising collecting a sample of cerebrospinal fluid(CSF) from a patient receiving at least one medical treatment for PD,measuring a poly(ADP-ribose) (PAR) concentration in said CSF sample, andcomparing the PAR concentration in said patient to a PAR concentrationin at least one control sample.
 15. The method according to claim 14,wherein the control sample is a CSF sample collected from said patientat an earlier time period.
 16. The method according to claim 14, whereinthe control sample is a CSF sample collected from a healthy donor. 17.The method according to claim 15, wherein if the PAR concentration insaid patient is higher than the PAR concentration in the control sample,then the medical treatment is not effective for said patient, and if thePAR concentration in said patient is the same or lower than the PARconcentration in the control sample, then the medical treatment iseffective for said patient.
 18. The method according to claim 14,wherein the medical treatment is administering to said patient one ormore pharmaceutical agents, performing surgery on said patient or acombination thereof.
 19. The method according to claim 14, whereinmeasuring the PAR concentration in said CSF sample is by sandwich ELISA.20. A method of diagnosing a patient with Parkinson's disease, saidmethod comprising collecting a sample of cerebrospinal fluid (CSF) froma patient, measuring a poly(ADP-ribose) (PAR) concentration in said CSFsample, and comparing the PAR concentration in said patient to a PARconcentration in at least one control sample.
 21. The method accordingto claim 20, wherein the control sample is a CSF sample collected fromsaid patient at an earlier time period.
 22. The method according toclaim 20, wherein the control sample is a CSF sample collected from ahealthy donor.
 23. The method according to claim 21, wherein if the PARconcentration in said patient is higher than the PAR concentration inthe control sample, then said patient is at high risk for developing PDor has PD, and if the PAR concentration in said patient is the same orlower than the PAR concentration in the control sample, then saidpatient is not at risk for developing PD or does not have PD.
 24. Atheranostic method for Parkinson's disease, said method comprisingcollecting a sample of cerebrospinal fluid (CSF) from a patient who isreceiving at least one medical treatment for PD, measuring apoly(ADP-ribose) (PAR) concentration in said CSF sample, comparing thePAR concentration in said patient to a PAR concentration in at least onecontrol sample.
 25. The theranostic method according to claim 24,wherein the control sample is a CSF sample collected from said patientat an earlier time period.
 26. The theranostic method according to claim24, wherein the control sample is a CSF sample collected from a healthydonor.
 27. The theranostic method according to claim 25 wherein if thePAR concentration in said patient is higher than the PAR concentrationin the control sample, then the method further comprises altering atleast one medical treatment received by said patient, and if the PARconcentration in said patient is the same or lower than the PARconcentration in the control sample, then the method further comprisesnot altering the medical treatment received by said patient.
 28. Themethod according to claim 24, wherein measuring the PAR concentration insaid CSF sample is by sandwich ELISA.